Detection of a putative hemolysin operon, hhdBA, of Haemophilus parasuis from pigs with Glasser disease

435805 JVDXXX10.1177/1040638711435805Assav acheep et al.Detection of a putative hemolysin operon Detection of a putative hemolysin operon, hhdBA, of Haemophilus parasuis from pigs with Glässer disease Journal of Veterinary Diagnostic Investigation 24(2) 339–343 © 2012 The Author(s) Reprints and permission: sagepub.com/journalsPermissions.nav DOI: 10.1177/1040638711435805 http://jvdi.sagepub.com Pornchalit Assavacheep,1 Anongnart Assavacheep, Conny Turni Abstract. The aim of the current study was to investigate whether polymerase chain reaction amplification of 16S ribosomal (r)RNA and a putative hemolysin gene operon, hhdBA, can be used to monitor live pigs for the presence of Haemophilus parasuis and predict the virulence of the strains present. Nasal cavity swabs were taken from 30 live, healthy, 1- to 8-week-old pigs on a weekly cycle from a commercial Thai nursery pig herd. A total of 27 of these pigs (90%) tested positive for H. parasuis as early as week 1 of age. None of the H. parasuis–positive samples from healthy pigs was positive for the hhdBA genes. At the same pig nursery, swab samples from nasal cavity, tonsil, trachea, and lung, and exudate samples from pleural/peritoneal cavity were taken from 30 dead pigs displaying typical pathological lesions consistent with Glässer disease. Twenty-two of 140 samples (15.7%) taken from 30 diseased pigs yielded a positive result for H. parasuis. Samples from the exudate (27%) yielded the most positive results, followed by lung, tracheal swab, tonsil, and nasal swab, respectively. Out of 22 positive samples, 12 samples (54.5%) harbored hhdA and/or hhdB genes. Detection rates of hhdA were higher than hhdB. None of the H. parasuis–positive samples taken from nasal cavity of diseased pigs tested positive for hhdBA genes. More work is required to determine if the detection of hhdBA genes is useful for identifying the virulence potential of H. parasuis field isolates. Key words: Glässer disease; Haemophilus parasuis; hhdBA; pigs. Haemophilus parasuis is a major bacterial pathogen, causing polyserositis, polyarthritis, and meningitis—Glässer disease.16 To date, at least 15 serovars have been recognized with a varying ability to induce severe disease. Serovars 1, 5, 10, and 12–14 are the most virulent serovars.6 Haemophilus parasuis culture is routinely used for diagnosis. The bacteria require V-factor (nicotinamide adenine dinucleotide) to grow on culture media. Bacterial culture from clinical samples is often difficult.11,19 In addition to bacterial culture, a polymerase chain reaction (PCR) for H. parasuis has been developed targeting the 16S ribosomal (r)RNA gene and has been shown to be capable of detecting 102colony forming units of H. parasuis.14 However, the PCR gives weak false-positive reactions for Actinobacillus indolicus.14 In 2007, a PCR was developed for the detection of H. parasuis also targeting the 16S rRNA gene.1 The PCR did not give any detectable PCR product for any other related bacterial species in the nicotinamide adenine dinucleotide–dependent family Pasteurellaceae, including A. indolicus, Actinobacillus porcinus, and Actinobacillus minor, but was not as sensitive as the previous study.1,14 An improved sensitivity of PCR over bacterial culture has been reported when using the newly developed real-time PCR.21 The fact that H. parasuis is a commensal bacterium, found in the upper respiratory tract of clinical healthy pigs,20 restricts the use of the 16S rRNA PCR as a diagnostic tool.14 However, identification of virulence genes of H. parasuis could be a useful method to indicate the virulence potential of detected isolates. Although numerous studies have attempted to identify virulence factors,4,5,7,9,10,12,18,23 information on virulence determinants of H. parasuis is still limited. Several reports have documented potential roles of virulence factors for fimbriae,13 transferrin-binding proteins,2 neuraminidase (sialidase) activity,8 TolC, disulfide interchange protein precursor DsbA and Dsbc and autotransporter adhesins,24 trimeric autotransporters,15 and outer membrane proteins.3,17,22 A 2009 report suggested that a putative hemolysin gene operon, hhdBA, which has only been found in virulent reference serovars, might be a novel potential virulence factor of H. parasuis.18 Therefore, the objective of the current study was to detect H. parasuis 16S rRNA and hhdBA genes in the nasal cavity of live clinically healthy pigs and in a variety of pathological lesions from nursery pigs at a nursery experiencing a Glässer disease outbreak. From Departments of Veterinary Medicine, Faculty of Veterinary Science, Chulalongkorn University, Pathumwan, Bangkok, Thailand (P Assavacheep), Animal Husbandry, Faculty of Veterinary Science, Chulalongkorn University, Pathumwan, Bangkok, Thailand (A Assavacheep), and The University of Queensland, Queensland Alliance for Agriculture and Food Innovation, Dutton Park, Queensland, Australia (Turni). 1 Corresponding Author: Pornchalit Assavacheep, Department of Veterinary Medicine, Faculty of Veterinary Science, Chulalongkorn University, Pathumwan, Bangkok 10330, Thailand. Pornchalit.A@chula.ac.th 340 Assavacheep et al. The pigs in the present study were from the nursery section of a pig farm in Ratchaburi, Thailand, previously diagnosed with an outbreak of Glässer disease. Prior to this investigation, necropsies were performed on 3 dead nursery pigs. Gross lesions included fibrinous pleuritis, pericarditis, and polyserositis. Haemophilus parasuis was cultured from these pathological lesions. Nasal swabs from 30 live, clinically healthy piglets were obtained in the first week of life, and weekly until 8 weeks of age. During these weekly early morning visits to the farm, necropsies were performed on any dead nursery pigs. Swab samples from 30 dead pigs were taken from the following sites: nasal turbinate, tonsil, trachea (middle part), and cut lung surface (both diaphragmatic lobes). When available, a transudate sample (approximately 1 ml) from pleural and/or peritoneal cavity was collected in a sterile microcentrifuge tube. All clinical samples were packed and stored on ice in polystyrene boxes and submitted to the laboratory on the same day of the farm visit. Bacterial genomic DNA was prepared from all clinical samples (swab or fluid), or from H. parasuis reference strains (serovars 1–15: NR4, SW140, SW114, SW124, Nagasaki, 131, 174, C5, D74, H367, H465, H425, IA-84-17975, IA-8422113, and IA-84-15995, and type strain CCUG 7984). To each swab sample, 400 µl of sterile phosphate buffered saline (PBS; pH 7.4) was added and centrifuged at 8,500 × g for 2 min. Suspensions were boiled at 100°C for 5 min and then cooled to room temperature. Tubes were centrifuged at 8,500 × g for 2 min. The supernatant was collected into a new microcentrifuge tube and frozen at –20°C until use. One 50-µl aliquot from pleural and peritoneal fluid was added to 350 µl of sterile PBS and boiled as described above. The supernatant from these samples was stored at –20°C until used. Genomic DNA from reference strains was used in validating a modification of a previously described PCR method.14 The DNA was prepared from a 1-µl loopful of the bacteria suspended in 100 µl of sterile water. The suspension was heated at 98°C for 5 min, followed by cooling on ice for 5 min. After centrifugation for 5 min at 17,380 × g, the supernatant was collected and stored at –20°C. Genomic DNA for the related species was prepared with a commercial kit,a per manufacturer’s instruction. A conventional PCR targeting the nuclear rRNA 16S rRNA gene with universal bacteria primers (5′-GAGTTTGATCCTGGC TCAG-3′ and 5′-AAGGAGGTGWTCCARCC-3′) was performed on the template of all bacterial species other than H. parasuis to confirm that the template was suitable for use in PCR. The related species used were Actinobacillus equuli (CCUG 2401), A. indolicus (CCUG39029), A. minor (CCUG 38923), A. porcinus (CCUG38924), A. rossi (CCUG12395), Actinobacillus suis (CCUG11624), Escherichia coli (BR316) (field strain), Haemophilus parainfluenzae (NCTC7857), Mannheimia haemolytica (CCUG408), Mannheimia varigena (CCUG38462), Pasteurella aerogenes (CCUG9995), Pasteurella canis (NCTC11621), Pasteurella langaaensis (NCTC11411), Pasteurella mairii (CCUG27189), Pasteurella multocida (NCTC10322), Pasteurella stomatis (NCTC11623), Pasteurella species B (SSIP683), Streptococcus suis (CCUG7984), Erysipelothrix rhusiopathiae (CCUG221), and Salmonella enterica serovar Typhimurium (field isolate; BR 258). Except where noted, all of the above were the taxonomic type or recognized reference strains. The PCR amplification of the 16S rRNA gene of H. parasuis was performed as previously described.14 The reaction consisted of 2 µl of boiled template DNA, 0.3 µM HPSforward primer, 0.3 µM HPS-reward primer, 7.5 µl of nuclease-free water, and 12.5 µl 2 × PCR master mixb (0.05 U/µl Taq DNA polymerase, 4 mM MgCl2 and 0.4 mM of each deoxyribonucleotide triphosphate [dNTP]). A total of 30 cycles of PCR amplification consisted of a denaturation step at 94°C for 30 sec, an annealing step at 52°C for 30 sec, and an extension step at 72°C for 2 min. The expected PCR product size was 821 bp. The duplex PCR amplification of hhdBA genes of H. parasuis was performed as previously described,18 with a modification of PCR compositions, which consisted of 2 µl of boiled template DNA, 0.3 µM MP_B1 and 0.3 µM MP_ A1 (forward), 0.3 µM MP_B2 and 0.3 µM MP_A2 (reverse), 6 µl of nuclease-free water, and 10 µl of 2× Prime Taq Premixc (10 µl composition consists of Prime Taq DNA polymerase 1 unit, Tris–HCl [pH 9.0], PCR enhancer, [NH4]2SO4, 4 mM MgCl2, enzyme stabilizer, sedimentation chemicals, loading dye, and 2 mM dNTP mixture). A denaturation step was performed at 94°C for 3 min, followed by a total of 32 cycles of PCR amplification consisting of another denaturation step at 94°C for 30 sec, an annealing step at 55°C for 1 min, an extension step at 72°C for 90 sec, and a final extension step of 72°C for 10 min. The expected PCR product sizes for hhdA and hhdB were 964 and 557 bp, respectively. The modified version of the published 16S rRNA-based, H. parasuis–specific PCR14 used in the present study was validated for specificity. All H. parasuis reference strains could be amplified with the modified PCR conditions (Table 1). The template of the 20 nontarget species was confirmed suitable for use in PCR amplification but did not amplify with the modified species-specific PCR.14 In the current study, the 16S rRNA-based, species-specific PCR was used to detect H. parasuis in nasal cavities of 30 live, clinically healthy pigs from 1 to 8 weeks of age, and from a variety of sites and pathological lesions from 30 diseased pigs, all sourced from a farm suffering a confirmed Glässer disease outbreak. Positive samples were then subjected to duplex PCR amplification of the hhdBA gene. The PCR detection of 16S rRNA and hhdA and hhdB genes of H. parasuis yielded single bands of 821, 964, and 557 bp, respectively. Following use of the H. parasuis–specific PCR on swabs of the nasal cavities of 30 live, clinically healthy pigs, 27 of Detection of a putative hemolysin operon 341 Table 1. Polymerase chain reaction (PCR) detection of 16S ribosomal (r)RNA, and hhdA and hhdB genes of Haemophilus parasuis reference strains for serovars 1–15.* Reference strains (serovar) Figure 1. Number of Haemophilus parasuis–positive nasal swab samples from clinical healthy, live piglets aged 1–8 weeks detected by the 16S ribosomal RNA–based, species-specific polymerase chain reaction. the pigs (90%) tested positive. Haemophilus parasuis was detectable as early as 1 week of age. The positive rate rose from week 1 to week 3 and stayed around 70% for the remaining weeks). The hhdBA genes were not detected from samples from nasal cavity, the majority of which were previously shown to be H. parasuis positive (Fig. 1). The 16S rRNA-based, species-specific PCR was used in an attempt to detect H. parasuis in 30 nasal swabs, 26 tonsil swabs, 28 tracheal swabs, 30 lung swabs, and 26 transudate samples (total 140 samples) collected from 30 diseased pigs (Table 2). Out of 140, only 22 samples (15.7%) tested positive by PCR (Table 2). The highest detection rate of H. parasuis was found from transudate, followed by lung swab, tracheal swab, and tonsil swab and nasal swab, respectively. Only 12 of 22 H. parasuis PCR–positive samples (54.5%) from diseased pigs were positive for hhdA and/or hhdB genes. Among the sampling sites with lesions, transudate samples had the most positive samples for hhdA (7/7, 100%) and hhdB (4/7, 57%), respectively. This was followed by tonsil (1/2, 50% for hhdA gene; 1/2, 50% for hhdB gene), lung (3/7, 43% for hhdA gene; 2/7, 29% for hhdB gene), and tracheal swabs (1/4, 25% for hhdA gene; 1/4, 25% for hhdB gene; Table 1). The overall detection rates of hhdA (60% with a range of 25–100%) in the current study were higher than hhdB genes (40% with a range of 25–57%). The hhdBA genes were undetectable from the 2 samples taken from the nasal cavity that previously tested positive for H. parasuis by the 16S rRNA-based, species-specific PCR. The infrequent detection of the organism by PCR in all samples tested from diseased animals (7–27%) is not unusual. Low detection rates and difficulties diagnosing H. parasuis have been previously reported.20,21 PCR detection Virulence* 16S rRNA NR 4 (1) SW 140 (2) SW 114 (3) SW 124 (4) Nagasaki (5) 131 (6) 174 (7) C5 (8) D74 (9) H367 (10) H465 (11) H425 (12) IA-84-17975 (13) IA-84-22113 (14) SD-84-15995 (15) CU70† ++ + 0 + ++ 0 0 ± 0 ++ 0 ++ ++ ++ + ND Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive hhdA hhdB Negative Negative Negative Negative Positive Negative Negative Negative Positive Negative Positive Positive Positive Positive Negative Positive Negative Negative Negative Negative Positive Negative Negative Negative Positive Negative Positive Positive Positive Positive Negative Positive *++, +, ±, and 0 represent highly virulent, moderate, mild, and avirulent H. parasuis reference serovars, respectively (data according to Kielstein and Rapp-Gabrielson6). ND = virulence properties unknown. †CU70 is a Thai H. parasuis isolate from a clinical case. Table 2. Polymerase chain reaction (PCR) detection of 16S ribosomal (r)RNA, and hhdA and hhdB genes of Haemophilus parasuis from dead pigs with Glässer disease.* No. of PCR positive (%) Location Nasal cavity Tonsil Trachea Lung Transudate Total Total sample tested 16S rRNA hhdA hhdB 30 2 (7) 0 0 26 28 30 26 140 2 (8) 1/2 (50) 1/2 (50) 4 (14) 1/4 (25) 1/4 (25) 7 (23) 3/7 (43) 2/7 (29) 7 (27) 7/7 (100) 4/7 (57) 22 (15.7) 12/20 (60) 8/20 (40) *When the hhdBA PCR was performed on the reference strains, serovars 5, 9, and 11–14 gave positive results (Table 1). Numbers in parentheses are percentages. Only limited information is available on the virulence determinants of H. parasuis.4,15 A recent report suggested the existence of a putative hemolysin gene operon hhdBA, which has been found only in virulent reference serovars and could be a novel virulence factor for H. parasuis.18 In the current study, PCR detection of hhdBA genes from H. parasuis reference strains appeared to differ from the previous results (Table 1).18 Some virulent strains6 did not harbor hhdBA, but some avirulent strains6 did in the current study. In contrast to 342 Assavacheep et al. results from a previous study,18 the hhdBA genes were not detected in the pathogenic serovar 15 strain, but were detected in the nonpathogenic serovars 9 and 11. Both studies show highly virulent reference strains, serovar 1 and serovar 10, lacking the gene. The hhdBA genes were only detected in H. parasuis–positive samples from diseased pigs. All samples taken from live clinically healthy pigs were negative. These results from the field samples suggest that there is a potential to discriminate pathogenic and nonpathogenic strains with the hhdBA PCR. However, the confusing results from the reference strains and the lack of any positive samples from the nasal cavity of clinically affected pigs highlight the need for further studies to clarify if the hhdBA PCR is a useful tool to discriminate pathogenic and nonpathogenic strains. The detection rate of the hhdBA gene of H. parasuis was greater in exudate samples than other samples. In contrast, hhdBA genes were undetectable from all nasal swabs of 30 healthy and 2 diseased pigs. This supports earlier evidence that hhdBA is likely to be involved in the virulence of H. parasuis.18 However, a larger number of nasal swabs need to be examined to confirm this finding. In addition, serovar profiling of nasal isolates might help to confirm the virulence status of strains in the nasal cavity. Based on previous in silico analysis, hhdBA genes were proposed as 2 components of putative hemolysin/export system.18 The detection rates of hhdA in the current study were higher than hhdB genes, similar to the earlier study.18 Furthermore, all hhdB-positive samples were positive in the hhdA PCR, whereas only some hhdA PCR–positive samples carried hhdB gene. It is possible that detection of the hhdA gene might be a more consistent indicator than hhdB gene for determination of virulence potential of field H. parasuis isolates. Acknowledgements Part of the PCR work was conducted in the facilities of the Chulalongkorn University Centenary Academic Development Project and Innovation Center for Veterinary Biotechnology, Faculty of Veterinary Science. The authors express their sincere gratitude to Professor R. Thanawongnuwech for providing Thai H. parasuis strain CU70. Sources and manufacturers a. PrepMan® Ultra, Applied Biosystems, Foster City, CA. b. Fermentas UAB, Vilnius, Lithuania. c. Prime Taq Premix, GeNet Bio, South Korea. Declaration of conflicting interests The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. Funding The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was funded by the Chulalongkorn University Veterinary Science Research Fund RG10/2553. References 1. Angen O, Oliveira S, Ahrens P, et al.: 2007, Development of an improved species specific PCR test for detection of Haemophilus parasuis. Vet Microbiol 119:266–276. 2. Charland N, D’Silva, CG, Dumont RA, Niven DF: 1995, Contact-dependent acquisition of transferrin-bound iron by two strains of Haemophilus parasuis. Can J Microbiol 41:70–74. 3. Chu YF, Gao PC, Zhao P, et al.: 2011, Genotyping of Haemophilus parasuis isolated from northwest China using PCRRFLP based on ompA gene. J Vet Med Sci 73:337–343. 4. 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